Transcription by eukaryotic RNA Polymerases (Pols) is the ultimate target for many signal transduction and developmental pathways, and understanding the mechanism of transcription is a key to understanding the mechanism of gene regulation. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription factors, chromatin modifying factors, or general transcription factors can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms utilized by the transcription machinery to promote transcription by Pol II and Pol III. The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, molecular, and genetic methods to examine the structures and mechanisms of the transcription machinery directing RNA Pols II and III in S. cerevisiae. Using a newly developed method for mapping the structural arrangement of large complexes, the structure of the Pol II pre-initiation Complex and Open Complex will be determined. The structures of these complexes will be compared on different promoter types. Based on these results, models for the mechanism of the general factors in transcription initiation will be derived and these models will be tested using biochemical and genetic assays. Similar methods will be utilized to determine the arrangement of the Pol III general factor Brf1 within the Pol III transcription machinery, permitting comparison of conserved and non conserved mechanisms utilized by different nuclear Pols. Finally, the mechanism of two cyclin-dependent kinases that regulate transcription during elongation will be explored. Analog-sensitive versions of these kinases will be utilized to identify relevant kinase targets. In vitro and in vivo assays will be used to determine the mechanisms whereby kinase activity regulates transcription.